Download The Nitrogen Cycle and Nitrogen Fixation

The Nitrogen
Cycle and
Nitrogen Fixation
The biosynthetic pathways to amino acids and
nucleotides share a requirement for nitrogen.
Soluble, biologically useful nitrogen compounds
are scarce in natural environments, so amino acids,
ammonia and nucleotides are used economically
by most organisms.
How is nitrogen from the environment introduced
into biological systems?
The most abundant form of nitrogen is present in
air, which is 4/5 molecular N2.
Only a few species of organisms can convert
atmospheric nitrogen into forms useful for living
organisms.
Therefore, the metabolic processes of different
organisms function in an independent manner to
salvage and reuse biologically available nitrogen
in a vast Nitrogen Cycle.
The first step in the nitrogen cycle is the reduction
(Fixation) of atmospheric nitrogen by nitrogen-fixing
bacteria to ammonia.
Ammonia can be used by most organisms, however,
soil bacteria that derive their energy by oxidation of
NH3 to nitrite (NO2) and nitrate (NO3) are so abundant
that nearly all ammonia reaching the soil becomes
oxidized to nitrate. This process is called Nitrification.
Plants and bacteria can reduce nitrate to ammonia
by the action of nitrate reductases (Nitrate assimilation).
Ammonia so formed can be used for biosynthesis of
amino acids in plants, and then used by animals for
protein synthesis.
Microbial degradation of proteins after death of an
organisms returns ammonia to the soil, where
nitrifying bacteria convert it to nitrate and nitrite
again.
A balance is achieved between fixed nitrogen and
atomospheric nitrogen by bacteria that convert
nitrate to N2 under anaeobic conditions. In this
process, Denitrification, soil bacteria use NO3
rather than O2 as the final electron acceptor in a
series of reactions (like Ox. Phos.) that generates
a proton gradient for ATP synthesis.
Amino acids and
other N-containing
compounds.
Nitrate Assimilation
Reduction by some
anaerobic bacteria;
most plants
Denitrification
NO3-
N2
Nitrogen
fixation
+
NH4
Degradation by
animals and
bacteria
Rhizobium
Azotobacter
NO2Nitrification by
soil bacteria
Synthesis in plants
and bacteria
Nitrification by
soil bacteria
Nitrate Assimilation
Nitrate assimilation occurs in two steps: Two
electron reduction of nitrate to nitrite catalyzed by
nitrate reductase, and the six electron reduction of
nitrite to ammonia, catalyzed by nitrite reductase.
Nitrate assimilation is the predominant means by
which green plants, algae and many microorganisms
acquire nitrogen. The pathway accounts for over
99% of the inorganic nitrogen (nitrate or N2)
assimilated into organisms.
Nitrate Reductase
NO3- + 2H+ + 2e-
NO2- + H2O
Nitrate reductase passes electrons via a mini electron
transport chain from NADH to NO3- to reduce it to
NO2-.
NADH
NADH+
[SH
FAD
cytochrome
b557
MoCo]
NO3NO2-
Nitrite Reductase in Plants
NO2- + 8H+ + 6e-
NH4 + 2H2O
Nitrite reductase requires 6 electrons to reduce NO2- to
ammonia. These electrons are obtained from photosynthetically reduced ferredoxin (Fdred).
Light
6 Fdred
6 Fdox
[(4FeS)
siroheme]
NO2NH4+
Nitrite reductases in higher plants are found in the
chloroplast, where they have access to (Fdred).
Only a few species of microorganisms, all
prokaryotic, can fix atmospheric nitrogen.
Some are free living, and others live as
symbionts in the root nodules of leguminous
plants.
The first product is ammonia, and the
reduction of N2 to ammonia is a highly
exergonic process:
N2 + 3H2
2NH3; delta G=-33 KJ/mol
The N-N triple bond is very stable, and so nitrogen
fixation has a very high activation energy.
The overall reaction for biological nitrogen fixation:
N2 + 10H+ + 8e- + 16ATP
2NH4+ +16ADP + 16Pi + H2
Biological nitrogen fixation is carried out by a
highly conserved complex of proteins called the
nitrogenase complex that has two components,
dinitrogenase reductase and dinitrogenase.
Dinitrogenase reductase is a dimer of two identical
subunits. It contains a single Fe4-S4 center and can
be oxidized and reduced by one electron. It also has
two binding sites for ATP and hydrolyzes ATP during
electron transfer.
Dinitrogenase is a tetramer with two copies of two
different subunits. It contains both Fe and Mo, and its
redox centers contain a total of 2 Mo, 32Fe and 30S
per tetramer. 1/2 of the Fe and S are present as
Fe4-S4 centers. The remainder is a novel Fe-Mo
cofactor (FeMoCo). Some forms of the enzyme
contain vanadium instead of molybdenum.
Nitrogen fixation is carried out by a highly reduced
form of dinitrogenase, and it requries 8 electrons; six
for the reduction of N2 and two to produce one
molecule of H2.
Dinitrogenase is reduced by transfer of electrons from
dinitrogenase reductase, and the 8 electrons are
transferred one at a time, with the reduced reductase
binding and the oxidized reductase dissociating from
dinitrogenase in a cycle. This cycle requires the
hydrolysis of ATP.
The source of electrons varies, but is usually
ferredoxin or flavodoxin.
Reduced Ferredoxin or Flavodoxin
electron transfer
Dinitrogenase Reductase
electron transfer, ATP
hydrolysis
Dinitrogenase
electron transfer
NH4+
Both ATP hydrolysis and ATP binding bring about
conformational changes that overcome the high
activation energy. Two ATP are hydrolyzed per
electron transferred (16 ATP total).
Nitrogenase is very unstable in the presence of oxygen.
The reductase is inactivated in air, with a half-life of
30s. Dinitrogenase has a half-life of 10 min. in air.
Nitrogen fixing organisms cope with this problem
in a variety of ways.
Regulation of Nitrogen Fixation
ADP inhibits the activity of nitrogenase. As the
energy charge of the cell drops, nitrogen fixation
is blocked.
Ammonia represses the expression of the nif genes,
the genes that encode the proteins of the nitrogen
fixing system.
Mechanisms for Dealing with Oxygen Toxicity:
•Some organisms live in an anaerobic environment.
•Some aerobic organisms uncouple their electron transport
chains. This results in an increase in the rate of electron
transport and an increased consumption of oxygen.
•Some filamentous cyanobacteria produce specialized
cells called heterocysts that have a very thick wall that
prevents oxygen from diffusing into the cell.
•The root nodules of plants produce the protein
leghemoglobin. This protein binds oxygen with high
affinity and transfers the oxygen to the electron transport
system.